FRET Monitoring of a Nonribosomal Peptide Synthetase Elongation Module Reveals Carrier Protein Shuttling between Catalytic Domains

Abstract Nonribosomal peptide synthetases (NRPSs) employ multiple domains, specifically arranged in modules, for the assembly‐line biosynthesis of a plethora of bioactive peptides. It is poorly understood how catalysis is correlated with the domain interplay and associated conformational changes. We developed FRET sensors of an elongation module to study in solution the intramodular interactions of the peptidyl carrier protein (PCP) with adenylation (A) and condensation (C) domains. Backed by HDX‐MS analysis, we discovered dynamic mixtures of conformations that undergo distinct population changes in favor of the PCP‐A and PCP‐C interactions upon completion of the adenylation and thiolation reactions, respectively. To probe this model we blocked PCP binding to the C domain by photocaging and triggered peptide bond formation with light. Changing intramodular domain affinities of the PCP appear to result in conformational shifts according to the logic of the templated assembly process.


Gene overexpression and protein purification
Genes were expressed and purified as previously described, [1] using the E.coli EC100 (ΔEntD) strain. After purification and dialysis, the concentration of all constructs was determined through their absorption at 280 nm, using the theoretical extinction coefficient calculated by ProtParam. [2] The unnatural amino acid ONBY was incorporated using nonsense suppression. [3] The corresponding pEVOL plasmid was introduced together with the encoding plasmid into the E.coli expression strain. ONBY was added in a final concentration of 1 mM to the growth medium.

Chemical modification with AF555 and AF647 maleimides
Proteins (4 µM) in NRPS assay buffer (50 mM HEPES, 100 mM NaCl, 1 mM EDTA, 10 mM MgCl2) were incubated with 2 mM TCEP for 30 min at 18°C to reduce cysteine side chains. AF555 and AF647 maleimides stock solutions in DMSO (10 µM) were premixed at a 7 to 8 equivalent ratio and added directly to the protein solution. The labeling reaction was performed for 30 min at 18°C and quenched by addition of 2 mM DTT. The labeled proteins were added to a Ni-NTA column (200 µL column material), washed with 2 mL NRPS assay buffer, and afterwards eluted with NRPS assay buffer containing 250 mM imidazole. Fractions containing the labeled protein of interest were combined, post-translationally converted overnight on ice in the active form and then dialyzed in NRPS assay buffer.

Post-translational conversion of apo into holo proteins
Apo proteins were incubated in NRPS assay buffer with 2 mM TCEP, 10 mM MgCl2, 50 equivalents coenzyme A or desulfo coenzyme A (25 equivalents) and 0.02 equivalents phosphopantetheine transferase Sfp from Bacillus subtilis. The reaction mixture was incubated S4 on ice overnight, followed by removal of excess CoA, TCEP and MgCl2 by dialysis against NRPS assay buffer.

MS assay to detect thioester formation
The formation of the proline thioester by the unlabeled and labeled enzymes was followed by ESI-TOF-MS. For the reaction the enzyme (10 µM) in NRPS assay buffer was incubated at 25°C with 2 mM each of ATP and L-proline. At respective time points the reaction was quenched by adding 45 µL of reaction mixture to 5 µL of a 10% formic acid solution. Samples were centrifuged (10 min, 15000 rpm, 4°C), 30 µL were transferred to mass vials and directly measured to prohibit precipitation. The mass analysis was performed as described earlier. [4]

FRET measurements
The measurements were basically performed as previously outlined. [4] FRET measurements were performed using either a 384-well plate (Greiner) together with the Tecan INFINITE M1000 Pro Microplate Reader (Tecan Austria GmbH) or a quartz cuvette (Hellma) together with the FP-8300 Fluorescence Spectrometer (Jasco Deutschland GmbH). As direct excitation wavelength 520 nm was chosen for AF555 maleimide and 650 nm for AF647 maleimide. The respective emission wavelengths were detected at 570 nm for AF555 and 674 nm for AF647. For INFINITE M1000 Pro and for FP-8300 a monochromator for both excitation as well as emission with a bandwidth of 5 nm was applied. With this setup for each measurement the fluorescence of both AF555 and AF647 after direct excitation was recorded as well as the emission of AF647 after AF555 excitation (energy transfer). For endpoint measurements (INFINITE M1000 Pro) data were taken after 30 min incubation with substrates or ligands, whereas for time-resolved measurements (FP-8300) readings were taken every minute. The sensors were used at a concentration of 300 nM (based on the acceptor fluorophore concentration) in a total volume of 50 µL assay buffer (pH 7) and incubated for 5 to 10 min until a stable fluorescence signal was recorded. Subsequently substrates or ligands were added as indicated in the figures (ATP and L-Pro at 2 mM, PPi at 10 mM, PPase at 0.2 U). The obtained raw data were normalized using the emission of AF647 after its direct excitation to eliminate fluorophore fluctuations based on varying enzyme concentrations. Additionally, the recorded energy transfer was corrected for the donor bleed-through and the crosstalk. The bleed-through was treated as a fraction of AF555 emission at 674 nm (6.66% for INFINITE M1000 Pro and 0.22% for FP-8300). The crosstalk designates the direct excitation of AF647 at 520 nm, calculated by excitation of the standalone dye, and was determined as a fraction of the observed fluorescence after excitation (5.38% for INFINITE M1000 Pro) and 1.41% for FP-8300). [5] The FRET ratio was calculated of the corrected data and normalized to 1 as the time point before substrate addition. Each experiment was performed with three biological replicates each containing multiple technical repeats. The mean and SD of each data sets were calculated and clustered. The combined SDs ( ) were calculated using , using as mean of individual and as means of combined data sets.

D-Phe-L-Pro-diketopiperazine (DKP) formation assay
The DKP formation assay was basically performed as previously described.
[6] 5 µM of fulllength holo-TycB1 was incubated with 5 mM ATP in assay buffer (pH 7.0). The reaction was started by adding 1 mM L-Pro, 1mM L-Phe, 10 mM MgCl2 and 0.5 µM of the partner initiation module holo-GrsA out of a master mixture, incubated at 37°C and quenched after 30 min by addition of 400 µL n-butanol/chloroform (4:1 v/v) and 200 µL ddH2O. The organic phase was extracted and the aqueous phase washed with 400 µL of the organic mixture. The organic phases were combined and afterwards washed twice with 300 µL ddH2O. After each washing the solutions were vortexed for 20 sec and centrifuged (2 min, 13000 rpm, 25°C) to separate the phases. The resulting organic phase was dried under vacuum and the remaining resolved in 30 µL of HPLC starting conditions (95% ddH2O, 5% acetonitrile and 0.1% trifluoroacetic acid). The solution was analyzed via HPLC (reversed-phase C18 column) and the area under the curve of the 210 nm UV/vis signal trail was used for the quantification of DKP formation.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS)
50 µM holo wildtype TycB1 was incubated for 30 minutes at room temperature either without any ligand (setting i) or in presence of 1 mM of each ATP and L-Pro (setting ii). The following sample preparation and measurement was performed as previously reported. [4,7] Source data for HDX-MS experiments are contained in the Supplemental Dataset, which also contains the further analysis with respect to differences in sequences stretches (seq) between the settings i and ii of holo wildtype TycB1. In short, the difference in deuterium incorporation between setting ii and that of setting i was calculated for each peptide and HDX timepoint. From that, the sum of differences over all sampled timepoints was calculated. In order illustrate the data for all peptides (out of which there are multiple overlapping ones, see Fig. S9) in a single plot, a peptide midpoint was calculated for each by addition of the first and last residue constituting the peptide followed by division with 2. Plotting the sum of the differences versus the peptide midpoints yields Fig. S11. [8] In order to define the boundaries between a peptide exhibiting no difference in HDX and one that shows HDX reduction, the peptide midpoints of these peptides were summed and divided by 2; a similar procedure was applied to define the boundaries between peptides exhibiting HDX differences of dissimilar amplitude (compare to Fig. 4A). This procedure results in sequence stretches (seq) that may differ in length from the length of the peptides, i.e., seq's may be longer than individual peptides in cases where multiple consecutive peptides exhibit a significant difference in HDX between settings ii and i (for example seq16, Fig. S10), or seq's may be shorter than individual peptides in cases where overlapping peptides were differently affected in HDX of setting ii versus i (for example seq5). Table S1. List of recombinantly produced proteins used in this study and their encoding plasmids, all generated in this study except pGV196. [6b] Name of construct* Encoding plasmid Vector backbone

SUPPLEMENTARY TABLES
* X denotes amino acid incorporation in response to amber stop codon.      [4,9] we constructed a new GrsA A-PCP control sensor. This new GrsA A-PCP sensor served as a direct comparison to our new TycB1 C-A*-PCP* and A*-PCP* sensors. To rule out or minimize possible effects of the nature and localization of the donor and acceptor fluorophores on the FRET output, the new GrsA A-PCP sensor was designed in the same way as the respective TycB1 sensors. Instead of a synthetic fluorophore conjugated to N152C and a C-terminally fused eGFP in our previous GrsA A-PCP sensor designs, [4,9] we here used stochastic bioconjugation of two cysteines, K125C and a Cterminally appended cysteine in the GVCTE sequence, to attach AF555 and AF647 via maleimide chemistry. K125 was determined as the similarly positioned residue compared to A553 in TycB1 based on a sequence alignment of the GrsA and TycB1 sequences. Likewise, the GVCTE sequence was appended to the respective PCPs in both cases at the same position based on the sequence alignment. (C) and (E) Scheme of the new GrsA A-PCP sensor. (D) Time-dependent change of the FRET ratio as determined for previous GrsA A-PCP sensor designs. [4,9] (F) Positions of N152C and K125C mapped on the structure of GrsA A-PCP.

S7
Together, these controls suggest that the new GrsA A-PCP FRET sensor design gives a qualitatively very similar FRET read-out as the previous sensor designs. Furthermore, they suggest that differences in the FRET read-out observed for the A-PCP didomain units of the GrsA and TycB1 sensors are not due to the FRET sensor design in terms of the nature of the fluorophores and the attachment positions of the fluorophores. Rather, differences in FRET read-out are likely to stem from structural differences between GrsA and TycB1 A-PCP unit, e.g. in the positioning and orientation of the PCP relative to the A N subdomain within a certain conformation like the transfer conformation, as well as from possible differences in relative population of individual conformations in the overall conformational equilibrium. Slight differences in positioning and orientation of the PCP relative to the A N subdomain could for example result in a high-FRET out-put for the Ppant-threading conformation, which is induced by PPi at excess concentrations in the P2 levels, whereas this conformation does not result in a high-FRET constellation in the GrsA A-PCP sensors. [4]   These experiments were performed to address differences observed in the interplay of the A-PCP didomain units of the new TycB1 sensors compared to our previously reported GrsA A-PCP sensors. To address the qualitatively opposite response to PPi in the P2 plateaus, we performed the assays with 2 mM PPi as an alternative concentration as shown in (A). Both 2 and 10 mM PPi showed a qualitatively similar effect. To verify that the high concentrations of Pi generated in the PPase reaction had no decisive impact on our observations, the control experiments in (B) were performed. Again, no significant changes were observed, ruling out the possibility that generated Pi could have significantly influenced the read-out of the FRET sensors. Similar findings were made for the control experiments in (C). These experiments show that PPase itself, without any high concentrations of added PPi that would be converted to Pi, does only have a negligible effect on the FRET ratio changes. However, the PPase addition obviously accounted for the differences observed between the P1 and P3 levels. This finding explains why no full reversibility back to the FRET ratio of plateau P1 is observed after PPase addition. Figure S9. PPi-induced conformational change to the Ppant-threading conformation. Previous work has suggested the existence of at least one intermediary conformation (I conformation) in the domain alternation mechanism between A and T conformations of the A domain. [4] The Ppant-threading conformation was proposed to be an intermediate for the prosthetic group of the PCP to enter and leave the A domain's active site. [4] The biochemical and structural requirements for the Ppant-threading conformation are well represented in an NRPS crystal structure showing the A N and A C subdomains of the A domain in a half-open conformation with the acylated Ppant-PCP bound to the active site. [10] In this structure (pdb code 5u89), [10] a binding site for PPi is plausible between the A N and A C subdomains. [4] By addition of PPi at high concentrations (e.g., 2 to 10 mM) this site is likely occupied, resulting in a conformational change from the transfer conformation to the Ppant-threading conformation, [4] as depicted in the Figure. Similar to the transfer conformation of the A-PCP didomain ensemble, the aminoacylated PCP remains bound to the A N subdomain, consistent with a high FRET ratio for the C-A*-PCP* and A*-PCP* sensors described in Figure 3. The Ppant-threading conformation can thus explain the high FRET ratio of plateau P2 for these sensors in their aminoacylated holo-forms.  [11] are highlighted in red. The color code of the background refers to the different domains and tag of the protein. Figure S11. HDX of representative peptides located in the sequence stretches 1-22 (seq1 -seq22), exemplifying the difference in HDX of full-length holo TycB1 between the buffer control (setting i, red trace) and upon incubation with with ATP and L-Pro (setting ii, blue trace). The TycB1 residue range covered by the sequences stretches (in brackets) was determined from overlapping peptides as described in the methods section. One representative peptide for each sequence stretch and its residue range is given except for very long sequence stretch 16, for which two representative peptides are depicted. Data represent the mean ± SD (n=3). Figure S12. Differences in HDX of holo TycB1 between the settings: buffer control (i) and ATP + L-Pro (ii). Graph depicts the total difference in HDX of setting ii minus setting i. To calculate the total HDX difference, the differences in HDX between settings ii and i were calculated for each time point of incubation in deuterated solvent (i.e. 10, 30, 95, 1000, 10000 s) and then summed up. The graphs depict the mean ± SD (n=3) of the total HDX difference according to the midpoint of the peptides (calculated as sum of the number of the first and last amino acid of the peptide divided by two). The dashed lines indicate the limits of the 95% confidence interval. Numbers denote the sequence stretches (seq) also shown in Figure S10 and Figure S11, which exhibit alterations in HDX above the confidence interval threshold. Sequence of TycB1(wt) modeled into the PDB structure 4ZXI, [12] in which the PCP domain is binding the C domain, using Phyre2. [13] Shown is the Vshaped structure of the C domain (grey) with interactive PCP domain (green). ONBY positions H35 (magenta) and Y337 (red) as well as the catalytically relevant H147 (blue) of the HHxxxDG motif are highlighted. S1007 with the Ppant moiety (orange) is also in sticks representation. The A domain is not shown for reasons of clarity. The H35 side chain points to the Ppant moiety, while the Y337 side chain points towards the body of the PCP domain. The photo-labile protecting group of ONBY is expected to result in steric clashes in both cases when the PCP were to productively bind the C domain. As the H35 position is situated closer to the active site than the Y337 position, the respective ONBY mutation might have a more pronounced effect on catalysis once the enzyme is in the C conformation, either by better sterically blocking the Pro-S-Ppant from the active site or by structurally perturbing the active site residues. (B) Close-up view.